BRPI0805560B1 - production process of an enzymatic preparation for cellulose hydrolysis of lignocellulosic waste and its application in ethanol production - Google Patents

production process of an enzymatic preparation for cellulose hydrolysis of lignocellulosic waste and its application in ethanol production Download PDF

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BRPI0805560B1
BRPI0805560B1 BRPI0805560A BRPI0805560A BRPI0805560B1 BR PI0805560 B1 BRPI0805560 B1 BR PI0805560B1 BR PI0805560 A BRPI0805560 A BR PI0805560A BR PI0805560 A BRPI0805560 A BR PI0805560A BR PI0805560 B1 BRPI0805560 B1 BR PI0805560B1
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production
hydrolysis
lignocellulosic
cellulose
waste
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BRPI0805560A
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Absai Da Conceição Gomes
Aline Machado De Castro
Claudia Julia Groposo Silveira
Danuza Nogueira Moyses
Emerson Pires Menezes
Luiz Fernando Martins Bandeira
Lídia Maria Melo Santa Anna
Nei Junior Pereira
Roberto Nobuyuki Maeda
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Petroleo Brasileiro Sa
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Priority to BRPI0805560A priority Critical patent/BRPI0805560B1/en
Priority to PT2009000343A priority patent/PT2010069018W/en
Priority to EP09795798.9A priority patent/EP2373787B1/en
Priority to PCT/GB2009/002929 priority patent/WO2010076552A1/en
Priority to DK09795798.9T priority patent/DK2373787T3/en
Priority to US13/002,171 priority patent/US20110250652A1/en
Publication of BRPI0805560A2 publication Critical patent/BRPI0805560A2/en
Priority to CO11090089A priority patent/CO6390008A2/en
Publication of BRPI0805560B1 publication Critical patent/BRPI0805560B1/en

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Abstract

processo de produção de um preparado enzimático para hidrólise de celulose de resíduos lignocelulósicos e sua aplicação na produção de etanol o processo da presente invenção baseia-se na produção microbiana de enzimas realizada a partir do crescimento do fungo penicilllum funiculosum em meio de cultivo adaptado com substrato celulósico. o processo da invenção compreende especialmente o tratamento fermentativo de um substrato lignocelulósico por meio de um fungo especialmente adaptado, visando à obtenção de um preparado enzimático capaz de hidrolisar a celulose e a hemicelulose para a produção de etanol.The process of producing an enzymatic preparation for cellulose hydrolysis of lignocellulosic residues and its application in ethanol production The process of the present invention is based on the microbial production of enzymes performed from the growth of the penicilllum funiculosum fungus in substrate-adapted culture medium. cellulosic. The process of the invention comprises especially the fermentative treatment of a lignocellulosic substrate by means of a specially adapted fungus to obtain an enzymatic preparation capable of hydrolyzing cellulose and hemicellulose for the production of ethanol.

Description

PROCESSO DE PRODUÇÃO DE UM PREPARADO ENZIMÁTICO PARA HIDRÓLISE DE CELULOSE DE RESÍDUOS LIGNOCELULÓSICOS E SUA APLICAÇÃO NA PRODUÇÃO DE ETANOLPROCESS FOR PRODUCTION OF AN ENZYMATIC PREPARATION FOR CELLULOSE HYDROLYSIS OF LIGNOCELLULOSTIC WASTE AND ITS APPLICATION IN ETHANOL PRODUCTION

CAMPO DA INVENÇÃOFIELD OF INVENTION

A presente invenção trata da produção de um preparado enzimático a partir de processo microbiológico, capaz de hidrolisar as frações hemicelulósicas e celulósicas das fibras de resíduos lignocelulósicos provenientes de ambientes florestais e agroindustriais. O objetivo deste processo hidrolítico é gerar altas concentrações de açúcares do tipo glicose e significantes concentrações de xilose que podem ser fermentadas por microrganismos com o objetivo de produzir etanol. FUNDAMENTOS DA INVENÇÃO O Brasil possui um excedente substancial de resíduos agroindustriais e agro-florestais sendo um dos maiores produtores de etanol e celulose do mundo. Por conseqüência, uma tecnologia que viabilize a produção de etanol a partir destes resíduos lignocelulósicos excedentes, além de contribuir para a solução de problemas ambientais, agregará valor a estes excedentes agroindustriais e agro-florestais, gerando vantagens econômicas para o país.The present invention is concerned with the production of an enzyme preparation from a microbiological process capable of hydrolyzing the hemicellulosic and cellulosic fractions of lignocellulosic waste fibers from forest and agroindustrial environments. The objective of this hydrolytic process is to generate high concentrations of glucose type sugars and significant concentrations of xylose that can be fermented by microorganisms for the purpose of producing ethanol. BACKGROUND OF THE INVENTION Brazil has a substantial surplus of agro-industrial and agro-forestry waste and is one of the largest producers of ethanol and cellulose in the world. Consequently, a technology that enables the production of ethanol from these surplus lignocellulosic residues, as well as contributing to the solution of environmental problems, will add value to these agroindustrial and agro-forestry surpluses, generating economic advantages for the country.

Para que se possa realizar o aproveitamento das frações polissacarídicas visando à produção de etanol é necessário hidrolisar estas frações eficientemente. O fracionamento desses polissacarídeos é efetuado através de um pré-tratamento, que consiste de uma reação conhecida como hidrólise ácida que objetiva a hidrólise da fração hemicelulósica. O sólido resultante desta etapa, rico na fração celulósica, ainda precisa ser tratado com o objetivo de remover a lígnina solúvel sob condições alcalinas para garantir a acessibilidade das enzimas à fibra celulósica. A conversão de celulose em etanol envolve dois passos fundamentais: hidrólise das longas cadeias das moléculas de celulose em açúcares (glicose) e fermentação desses açúcares em etanol. Na natureza, esses processos são encetados por fungos e bactérias, que secretam as enzimas capazes de hidrolisar a celulose (chamadas celulases), e principalmente por leveduras, quando se trata da fermentação dos açúcares em álcool. A maior dificuldade que precisa ser superada se refere ao próprio microrganismo, que precisa ser resistente às condições operacionais, principalmente no que tange às concentrações de inibidores gerados no meio reacional.In order to take advantage of the polysaccharide fractions aiming at ethanol production it is necessary to hydrolyze these fractions efficiently. The fractionation of these polysaccharides is performed through a pretreatment, which consists of a reaction known as acid hydrolysis that aims at hydrolysis of the hemicellulosic fraction. The solid resulting from this step, rich in the cellulosic fraction, still needs to be treated in order to remove the soluble lignin under alkaline conditions to ensure the accessibility of the enzymes to the cellulosic fiber. Converting cellulose to ethanol involves two key steps: hydrolysis of the long chains of cellulose molecules to sugars (glucose) and fermentation of these sugars in ethanol. In nature, these processes are initiated by fungi and bacteria, which secrete enzymes capable of hydrolyzing cellulose (called cellulases), and mainly by yeast, when it comes to the fermentation of sugars in alcohol. The biggest difficulty that needs to be overcome is the microorganism itself, which needs to be resistant to operating conditions, especially regarding the concentrations of inhibitors generated in the reaction medium.

Atualmente, um dos grandes gargalos para a produção bioquímica de etanol de segunda geração é a obtenção de um preparado enzimático de baixo custo e que apresente uma boa eficiência de hidrólise de polissacarídeos como a celulose (cujo monômero é a glicose) e hemicelulose (heteropolímero cujo monômero principal é a xilose). A obtenção dessas enzimas vem sendo alvo de intensa pesquisa pelo mundo, com participação de grandes empresas (inclusive petroleiras) e órgãos governamentais, e é fundamental para o desenvolvimento de tecnologias denominadas "limpas".Currently, one of the major bottlenecks for biochemical production of second generation ethanol is obtaining a low cost enzyme preparation that has good hydrolysis efficiency of polysaccharides such as cellulose (whose monomer is glucose) and hemicellulose (heteropolymer whose main monomer is xylose). Obtaining these enzymes has been the subject of intense research around the world, with the participation of large companies (including oil companies) and government agencies, and is fundamental for the development of technologies called "clean".

Entretanto, os altos custos dessas enzimas inviabilizam sua grande aplicação em escala comercial e impedem a implementação de plantas industriais.However, the high costs of these enzymes make their large-scale application unfeasible and prevent the implementation of industrial plants.

TÉCNICA RELACIONADA A produção de etanol por tecnologia biológica vem sendo estudada de há muito, porém nos últimos anos sofreu um grande impulso. O grande obstáculo a ser superado se refere â produtividade, ou seja, conseguir um processo economicamente viável e com bons rendimentos, utilizando matéria-prima com grande disponibilidade e baixo valor agregado. O documento de patente GB 2253633, que corresponde ao documento de patente brasileiro PI 9200100-9 de 15/01/92, descreve um processo para produzir etanol a partir de biomassa em que o substrato inclui um hidrolisado de celulose, hemicelulose e amido, visando produzir açúcares fermentáveis de seis carbonos. Para a fermentação é utilizada uma cepa de levedura geneticamente modificada (Brettanomyces custersii CBS 5512) que produz a enzima β-glucosidade, o que torna esta levedura com habilidade de fermentar tanto glicose quanto celobiose. No entanto, ainda permanece sem solução a fermentação de pentoses.RELATED TECHNIQUE The production of ethanol by biological technology has been studied for a long time, but in recent years has experienced a major boost. The major hurdle to be overcome is productivity, that is, achieving an economically viable process with good yields using highly available raw material and low added value. GB 2253633, which corresponds to Brazilian patent document PI 9200100-9 of 01/15/92, describes a process for producing ethanol from biomass wherein the substrate includes a cellulose, hemicellulose and starch hydrolyzate, aiming at produce six-carbon fermentable sugars. Fermentation uses a genetically modified yeast strain (Brettanomyces custersii CBS 5512) that produces the enzyme β-glucosity, which makes this yeast capable of fermenting both glucose and cellobiose. However, pentose fermentation remains unsolved.

Torget e colaboradores (US 5,705,369) descrevem um processo genérico de pré-hidrolise de materiais lignocelulósicos em que são investigadas diferentes combinações de faixas de temperatura e de tempo de reação, visando obter melhor percentual de separação de hemicelulose e lignina da celulose.Torget et al. (US 5,705,369) describe a generic process for prehydrolysis of lignocellulosic materials in which different combinations of temperature and reaction time ranges are investigated in order to obtain a better percentage of hemicellulose and lignin separation from cellulose.

Warzwoda e colaboradores (BR 0600409-1) descrevem um processo de produção de enzimas celulolíticas e hemicelulolíticas a partir de resíduos (madeiras de espécies folhosas e palhas de cereais). Tais resíduos são utilizados como fonte de carbono indutora para obtenção dessas enzimas, utilizando-se cepas de Trichoderma reesei geneticamente melhoradas e notadamente recombinadas. As cepas selvagens deste microorganismo têm a capacidade de secretar, em presença de um substrato indutor (a celulose, por exemplo), o complexo enzimático considerado o mais adaptado à hidrólise da celulose. Trata-se, portanto, de um processo para produção de enzimas celulolíticas e/ou hemicelulolíticas produzidas pela cepa especializada.Warzwoda et al. (BR 0600409-1) describe a process of producing cellulolytic and hemicellulolytic enzymes from residues (hardwood and cereal straw). Such residues are used as an inducing carbon source to obtain these enzymes using genetically improved and remarkably recombined Trichoderma reesei strains. Wild strains of this microorganism have the ability to secrete, in the presence of an inducing substrate (cellulose, for example), the enzyme complex considered to be the most suitable for cellulose hydrolysis. Therefore, it is a process for the production of cellulolytic and / or hemicellulolytic enzymes produced by the specialized strain.

Santa Anna e colaboradores (BR 0505299-8) ensinam um processo para produção de etanol visando o tratamento do resíduo sólido resultante da hidrólise ácida de bagaço de cana. Segundo este processo um hidrolisado da fração hemicelulósica do bagaço de cana, rico em xilose, foi obtido por meio da hidrólise branda com ácido sulfúrico diluído, e fermentado utilizando-se uma cepa da levedura Pichia stipitis adequadamente aclimatada ao substrato principal do hidrolisado (xilose). O resíduo sólido resultante da hidrólise ácida (celulignina) foi tratado no próprio reator para remoção de lignina, através de uma série de lavagens alcalinas a fim de deixar as fibras celulósicas aptas a receberem uma carga enzimática.Santa Anna et al. (BR 0505299-8) teach a process for ethanol production aiming at the treatment of solid residue resulting from acid hydrolysis of sugarcane bagasse. According to this process, a hydrolysate from the hemicellulosic fraction of sugarcane bagasse, rich in xylose, was obtained by gentle hydrolysis with dilute sulfuric acid and fermented using a properly acclimatized Pichia stipitis yeast strain to the main hydrolyzate substrate (xylose). . The solid residue resulting from acid hydrolysis (cellulignin) was treated in the lignin removal reactor itself through a series of alkaline washes to leave the cellulosic fibers able to receive an enzymatic charge.

Santa Anna e colaboradores (BR 0605017-4) descrevem um processo para obtenção de etanol a partir de materiais lignocelulósicos por via enzimática segundo o qual a fração hemicelulósica é submetida a uma hidrólise branda com ácido sulfúrico, e o material sólido resultante desta hidrólise, submetido ao processo de sacarificação (hidrólise enzimática) simultânea à fermentação alcoólica rápida, em condições tais que permitam aumentar significativamente a conversão em etanol em tempos muito reduzidos, utilizando altas concentrações de sólidos (15% a 25 %).Santa Anna et al. (BR 0605017-4) describe a process for enzymatically obtaining ethanol from lignocellulosic materials whereby the hemicellulosic fraction is subjected to a mild hydrolysis with sulfuric acid and the solid material resulting from this hydrolysis is subjected. the saccharification process (enzymatic hydrolysis) simultaneous with rapid alcoholic fermentation under conditions such as to significantly increase the conversion to ethanol at very short times using high solids concentrations (15% to 25%).

Chung e Day (WO 2008/095098) apresentam um processo para obtenção de açúcares a partir de biomassa lignocelulósica no qual se faz um pré-tratamento alcalino a quente do material com uma mistura de hidróxido de cálcio e água à temperatura de 80°C a 140°C durante cerca de 30 min a 3 horas. Após o tratamento, o bagaço é prensado; o líquido contém principalmente componentes solúveis de lignina, além de cal (que pode ser recuperada) e o material sólido fibroso é submetido à hidrólise por enzimas celulásicas. Segundo os autores, este tratamento modifica a estrutura lignocelulósica de modo que possa ser rapidamente solubilizada pela celulase, mesmo usando altos teores de sólidos (10% a 30%), sem que a atividade enzimática seja afetada. Foram utilizadas enzimas comerciais, como por exemplo, Spezyme CP (Genecor International Co) e Novo I88 (Novozyme). O objetivo do processo da presente invenção é oferecer um preparado enzimático contendo enzimas preparadas in situ (ou dedicadas) de baixo custo, capaz de viabilizar economicamente o escalonamento da tecnologia bioquímica para aplicação em processos de obtenção de etanol a partir de materiais lignocelulósicos. O preparado obtido pode ser aplicado ao sólido gerado após a hidrólise da fração hemicelulósica e também a processos de fermentação que utilizam sacarificação simultânea (SSF), como os mencionados acima.Chung and Day (WO 2008/095098) present a process for obtaining sugars from lignocellulosic biomass in which hot alkaline pretreatment of the material with a mixture of calcium hydroxide and water at a temperature of 80 ° C to 140 ° C for about 30 min to 3 hours. After treatment, the bagasse is pressed; The liquid contains mainly soluble lignin components as well as lime (which can be recovered) and the fibrous solid material is subjected to hydrolysis by cellulosic enzymes. According to the authors, this treatment modifies the lignocellulosic structure so that it can be quickly solubilized by cellulase, even using high solids contents (10% to 30%), without affecting enzymatic activity. Commercial enzymes such as Spezyme CP (Genecor International Co) and Novo I88 (Novozyme) were used. The object of the process of the present invention is to provide an enzyme preparation containing low cost in situ prepared (or dedicated) enzymes capable of economically scaling up biochemical technology for application in processes of obtaining ethanol from lignocellulosic materials. The obtained preparation can be applied to the solid generated after hydrolysis of the hemicellulosic fraction and also to fermentation processes using simultaneous saccharification (SSF), as mentioned above.

SUMÁRIO DA INVENÇÃO O processo da presente invenção baseia-se na produção microbiana de enzimas realizada a partir do crescimento do fungo em meio de cultivo adaptado com substrato celulósico. A invenção tem por objetivo a produção das enzimas celulásicas por via fermentativa utilizando o fungo Penicillium funiculosum, na qual podem ser usados substratos sintéticos (como o avicel ou carboximetilcelulose -CMC), ou ainda os próprios resíduos lignocelulósico e agroflorestais, in natura ou pré-tratados.SUMMARY OF THE INVENTION The process of the present invention is based on microbial enzyme production carried out from fungal growth in culture medium adapted with cellulosic substrate. The invention is directed to the production of cellulosic enzymes by fermentation using the fungus Penicillium funiculosum, in which synthetic substrates (such as avicel or carboxymethylcellulose -CMC) can be used, as well as the lignocellulosic and agroforestry residues themselves, in natura or pre- treated.

DESCRIÇÃO DETALHADA DA INVENÇÃO O processo da presente invenção compreende especialmente o tratamento fermentativo de substratos lignocelulósicos e agro florestais por meio de um fungo especialmente adaptado, visando à obtenção de um preparado enzimático capaz de hidrolisar a celulose e a hemicelulose para a produção de álcool. A invenção fundamenta-se na produção das enzimas celulásicas por via fermentativa utilizando o fungo Penicillium funiculosum ATCC 11797, podendo ser usados substratos sintéticos, ou os próprios resíduos lignocelulósicos e agros florestais, in natura ou pré-tratados. A etapa seguinte do processo compreende a concentração das enzimas produzidas na fermentação, utilizando sistemas de membrana -como um sistema combinado de microfiltração e ultrafiltração, ou por evaporação - por exemplo, rotaevaporador. Finalmente, adiciona-se a este concentrado de enzimas um aditivo, de maneira a melhorar a atuação das enzimas na quebra da celulose. Este aditivo compreende um biotensoativo do tipo glicolipídeo que promove o aumento da acessibilidade das enzimas à celulose e hemicelulose.DETAILED DESCRIPTION OF THE INVENTION The process of the present invention comprises especially the fermentative treatment of lignocellulosic and agro-forestry substrates by means of a specially adapted fungus to obtain an enzymatic preparation capable of hydrolyzing cellulose and hemicellulose for alcohol production. The invention is based on the production of cellulosic enzymes by fermentation using the fungus Penicillium funiculosum ATCC 11797, either synthetic substrates or the lignocellulosic residues and forest themselves, either fresh or pretreated. The next step of the process comprises concentrating the enzymes produced in the fermentation using membrane systems - such as a combined microfiltration and ultrafiltration system, or by evaporation - for example, rotary evaporator. Finally, an additive is added to this enzyme concentrate in order to improve the role of enzymes in cellulose breakdown. This additive comprises a glycolipid-like biotensive agent that promotes increased accessibility of enzymes to cellulose and hemicellulose.

Para melhor compreensão do invento, o processo pode ser descrito resumidamente da seguinte maneira: O fungo é colocado em contato com o substrato celulósico em um meio de cultivo adaptado por um período de quatro a sete dias, para que as enzimas sejam produzidas. Durante este tempo, o microrganismo libera para o meio de cultivo altas concentrações de proteínas com propriedades catalíticas, ou seja, enzimas celulásicas e hemicelulásicas, tais como xilanases, endoglucanases, exoglucanases e β-glucosidases, com a finalidade de quebrar as frações celulósicas e hemicelulósicas presentes no substrato. Após este período, o meio de cultivo contendo as enzimas é submetido a um processo de concentração pelo uso de membranas ou processos evaporativos, seguido de aditivação. O preparado enzimático (extrato) foi produzido com materiais residuais da produção convencional de etanol, como por exemplo, bagaço e palha de cana-de-açúcar, apresentando baixo custo de produção, permitindo que seja produzido in situ, tornando viável o escalonamento da produção de etanol de segunda geração.For a better understanding of the invention, the process can be briefly described as follows: The fungus is placed in contact with the cellulosic substrate in a culture medium adapted for a period of four to seven days for enzymes to be produced. During this time, the microorganism releases into the culture medium high concentrations of proteins with catalytic properties, ie cellulosic and hemicellulosic enzymes such as xylanases, endoglucanases, exoglucanases and β-glucosidases, in order to break down the cellulosic and hemicellulosic fractions. present in the substrate. After this period, the culture medium containing the enzymes is subjected to a concentration process by the use of membranes or evaporative processes, followed by additives. The enzymatic preparation (extract) was produced with waste materials from conventional ethanol production, such as bagasse and sugarcane straw, presenting low production cost, allowing it to be produced in situ, making production scheduling viable. of second generation ethanol.

Também pode ser utilizado como fonte de lignocelulose material agro florestal, como por exemplo, cavacos e resíduos da indústria de papel e celulose.It can also be used as a source of lignocellulose agro-forest material such as chips and waste from the pulp and paper industry.

Os Exemplos que se seguem são apresentados a título de ilustração apenas, não representando, todavia, qualquer tipo de limitação ao invento. EXEMPLO 1 Neste Exemplo pretende-se demonstrar a potencialidade de utilização do processo agora proposto para a obtenção do preparado enzimático.The following Examples are given by way of illustration only, but do not represent any limitation of the invention. EXAMPLE 1 In this Example we intend to demonstrate the potential use of the process now proposed for obtaining the enzyme preparation.

Utilizando o processo da invenção conforme descrito em detalhe anteriormente, foi possível produzir um extrato com altas concentrações de enzimas celulásicas, expressas em atividades enzimáticas (Ul/L) e concentrações protéicas (mg/L). A Tabela 1 a seguir apresenta o resultado da medida de atividade enzimática dos extratos ao final da fermentação e depois da concentração.Using the process of the invention as described in detail above, it was possible to produce an extract with high concentrations of cellulosic enzymes expressed as enzymatic activities (Ul / L) and protein concentrations (mg / L). Table 1 below presents the result of the enzyme activity measurement of the extracts at the end of fermentation and after concentration.

Esses extratos enzimáticos concentrados foram testados no processo de hidrólise (sacarificação) da celulose e hemicelulose contidas no bagaço de cana-de-açúcar pré-tratado, ou seja, submetido à hidrólise branda e lavagens com aquecimento para remoção da lignina, e os resultados estão na Tabela 2. A medida dos açúcares (glicose, xilose e celobiose) foi avaliada pelos métodos de cromatografia líquida (HPLC) e medida espectrofotométrica de açúcares redutores totais (ART). Nesta tabela, também são apresentados, a título de comparação, resultados obtidos com um preparado comercial (GENENCOR®), normalizada na mesma concentração enzimática por grama de celulose. A Tabela 2 apresenta as quantidades de glicose, proveniente da hidrólise da celulose. Observa-se também que, no extrato concentrado da invenção, há enzimas xilanásicas que podem converter a hemicelulose em xilose, que também poderá ser aproveitada para conversão a etanol. Isto comprova que o extrato enzimático produzido poder ser aplicado em processos de sacarificação e fermentação simultâneas (SSF) e sacarificação e co-fermentação simultâneas (SSCF). EXEMPLO 2 Este Exemplo tem por objetivo demonstrar a aplicabilidade do preparado enzimático, objeto da presente invenção, na hidrólise de resíduo lignocelulósico pré-tratado por hidrólise branda e lavagens com aquecimento para remoção da lignina, proveniente da produção convencional de etanol de cana-de-açúcar. O preparado enzimático foi aplicado em concentrações entre 5 e 30 FPU/g, em temperaturas entre 30°C e 60°C, durante 6 a 48 horas de sacarificação. A concentração de glicose liberada após o tratamento variou de 10 a 40 g/L).These concentrated enzyme extracts were tested in the hydrolysis (saccharification) process of the cellulose and hemicellulose contained in the pretreated sugarcane bagasse, ie, subjected to mild hydrolysis and heat washes to remove lignin, and the results are Table 2. The measurement of sugars (glucose, xylose and cellobiose) was evaluated by liquid chromatography (HPLC) and spectrophotometric measurements of total reducing sugars (ART). This table also presents, by way of comparison, results obtained with a commercial preparation (GENENCOR®), normalized to the same enzymatic concentration per gram of cellulose. Table 2 shows the amounts of glucose from cellulose hydrolysis. It is also noted that in the concentrated extract of the invention there are xylanase enzymes which can convert hemicellulose to xylose which can also be used for conversion to ethanol. This proves that the enzyme extract produced can be applied in simultaneous saccharification and fermentation (SSF) and simultaneous saccharification and co-fermentation (SSCF) processes. EXAMPLE 2 This Example is intended to demonstrate the applicability of the enzyme preparation object of the present invention in the hydrolysis of lignocellulosic residue pretreated by mild hydrolysis and heat washes to remove lignin from conventional sugarcane ethanol production. sugar. The enzyme preparation was applied at concentrations between 5 and 30 FPU / g at temperatures between 30 ° C and 60 ° C for 6 to 48 hours of saccharification. The glucose concentration released after treatment ranged from 10 to 40 g / L).

Os resultados alcançados nos permitem afirmar ser esta uma grande vantagem do extrato produzido pelo processo da invenção, por P. funiculosum, em relação ao preparado comercial avaliado, pois sinaliza que esse pool enzimático produzido pode ser aplicado em processos de sacarificação e co-fermentação simultâneas, que é uma forma de condução mais integrada e menos complexa em termos de equipamentos.The results obtained allow us to state that this is a great advantage of the extract produced by the process of the invention by P. funiculosum over the evaluated commercial preparation, since it indicates that this enzyme pool produced can be applied in simultaneous saccharification and co-fermentation processes. , which is a more integrated and less complex form of driving.

Outra vantagem do extrato de P. funiculosum é a elevada atividade β-glucosidásica, que permite a conversão de todo o dissacarídeo celobiose gerado em glicose (açúcar fermentável), enquanto que com o produto comercial permanece uma quantidade residual de celobiose, um dissacarídeo que não pode ser aproveitado para obtenção de etanol, no processo fermentativo.Another advantage of P. funiculosum extract is the high β-glucosidase activity, which allows the conversion of all cellobiose disaccharide generated to glucose (fermentable sugar), while with the commercial product remains a residual amount of cellobiose, a disaccharide that does not It can be used to obtain ethanol in the fermentation process.

Outra vantagem observada é a ação sinérgica das enzimas deste preparado, na hidrólise da celulose e hemicelulose quebrando esses polissacarídeos a monômeros de açúcares, e não gerando açúcares intermediários residuais não fermentáveis, que não são aproveitados para a produção de etanol.Another advantage observed is the synergistic action of the enzymes of this preparation, in the hydrolysis of cellulose and hemicellulose, breaking these polysaccharides to sugar monomers, and not generating non-fermentable residual intermediate sugars, which are not used for ethanol production.

Além disso, foi produzido a partir de resíduos agroindustriais ou agros florestais abundantes no Brasil, e aplicado in situ na hidrólise desses mesmos resíduos, inclusive resíduos com composições distintas, obtendo-se produtividade superior ou similar àquela resultante de produtos comerciais.In addition, it was produced from agroindustrial or agroforestry residues abundant in Brazil, and applied in situ in the hydrolysis of these same residues, including residues with different compositions, obtaining higher productivity or similar to that resulting from commercial products.

Outra grande vantagem desta invenção esta relacionada ao alto custo das enzimas celulásicas oferecidas no mercado, fato que até então eram impeditivos ao escalonamento da tecnologia bioquímica de conversão de materiais celulósicos para etanol. Esta invenção possibilita a produção destas enzimas “in situ” ou dedicadas, reduzindo fortemente o custo de produção das mesmas.Another major advantage of this invention is related to the high cost of cellulosic enzymes offered on the market, a fact that hitherto prevented the staggering of biochemical technology for converting cellulosic materials to ethanol. This invention enables the production of these enzymes in situ or dedicated, greatly reducing the cost of their production.

REIVINDICAÇÕES

Claims (6)

1- PROCESSO DE PRODUÇÃO DE UM PREPARADO ENZIMÁTICO PARA HIDRÓLISE DE CELULOSE DE RESlDUOS LIG NOCELULÓSICOS, caracterizado por compreender as etapas de: - colocar o fungo da cepa Penicillium funiculosum ATCC 11797 em contato com um substrato celulósico em um meio de cultivo adaptado por um período de quatro a sete dias, para que enzimas celulásicas e hemicelulásicas, tais como xilanases, endoglucanases, exoglucanases e p-glucosidases sejam produzidas; - submeter o meio de cultivo contendo as enzimas a um processo de concentração pelo uso de membranas ou processos evaporativos e aditivação; - aplicar o extrato enzimático produzido em processo de sacarificação e fermentação simultâneas (SSF), com hidrólise do resíduo lignocelulósico em açúcares fermentáveis e a sua fermentação de forma a gerar álcool.1- PRODUCTION PROCESS OF AN ENZYMATIC PREPARATION FOR CELLULOSE HYDROLYSIS OF NOCELLULOSIC CALL WASTE, characterized by the steps of: - placing the Penicillium funiculosum ATCC 11797 strain in contact with a cellulosic substrate adapted for a period four to seven days, for cellulosic and hemicellular enzymes such as xylanases, endoglucanases, exoglucanases and p-glucosidases to be produced; - subjecting the culture medium containing the enzymes to a concentration process by the use of membranes or evaporative processes and additives; - Apply the enzymatic extract produced in simultaneous saccharification and fermentation (SSF) process, with hydrolysis of lignocellulosic residue in fermentable sugars and their fermentation in order to generate alcohol. 2- PROCESSO DE PRODUÇÃO DE UM PREPARADO ENZIMÁTICO PARA HIDRÓLISE DE CELULOSE DE RESÍDUOS LIG NOCELULÓSICOS, de acordo com a reivindicação 1, caracterizado por o material lignocelulósico compreender o bagaço de cana-de-açúcar in natura ou pré-tratado, submetido à hidrólise branda e lavagens com aquecimento para remoção da lignina; e por o material agro florestal compreender cavacos e resíduos da indústria de papel.Process for the production of an enzyme preparation for cellulosic hydrolysis of non-cellulosic alloy residues according to claim 1, characterized in that the lignocellulosic material comprises fresh or pretreated sugarcane bagasse subjected to mild hydrolysis. and heat washes to remove lignin; and because the agro-forestry material comprises chips and waste from the paper industry. 3- PROCESSO DE PRODUÇÃO DE UM PREPARADO ENZIMÁTICO PARA HIDRÓLISE DE CELULOSE DE RESÍDUOS LIGNOCELULÓSICOS, de acordo com a reivindicação 1, caracterizado por o extrato enzimático produzido poder ser aplicado na hidrólise da celulose e hemicelulose quebrando esses polissacarídeos a monômeros de açúcares, e não gerando celobiose.Process for the production of an enzymatic preparation for cellulose hydrolysis of lignocellulosic waste according to claim 1, characterized in that the enzymatic extract produced can be applied in the hydrolysis of cellulose and hemicellulose by breaking down these polysaccharides to sugar monomers. Cellobiose. 4- PROCESSO DE PRODUÇÃO DE UM PREPARADO ENZIMÁTICO PARA HIDRÓLISE DE CELULOSE DE RESÍDUOS LIGNOCELULÓSICOS» de acordo com a reivindicação 1, caracterizado por o preparado enzimático ser produzido in situ, com materiais residuais da produção convencional de etanol de cana-de-açúcar.Process for the production of an enzyme preparation for cellulose hydrolysis of lignocellulosic waste according to claim 1, characterized in that the enzyme preparation is produced in situ with waste materials from the conventional production of sugarcane ethanol. 5- PROCESSO DE PRODUÇÃO DE UM PREPARADO ENZIMÁTICO PARA HIDRÓLISE DE CELULOSE DE RESÍDUOS LIGNOCELULÓSICOS, de acordo com a reivindicação 1, caracterizado por o aditivo utilizado ser um biotensoativo do tipo glicolipídeo que promove o aumento da acessibilidade das enzimas à celulose e hemicelulose,Production process of an enzyme preparation for cellulose hydrolysis of lignocellulosic residues according to claim 1, characterized in that the additive used is a glycolipid-like biotensive agent that promotes increased accessibility of enzymes to cellulose and hemicellulose. 6- PROCESSO DE PRODUÇÃO DE ETANOL POR HIDRÓLISE DE CELULOSE DE RESÍDUOS LIGNOCELULÓSICOS, caracterizado oor utilizar o preparado enzimático produzido in situ de acordo com a reivindicação 1, com materiais residuais in natura e pré-tratados da produção convencional de etanol de cana-de-açúcar, tais como bagaço e palha, e cavacos e materiais residuais da indústria de papel e celulose.6. A process for producing ethanol by cellulose hydrolysis of lignocellulosic waste, characterized in that it uses the in situ produced enzymatic preparation according to claim 1, with pre-treated in natura waste materials from conventional sugarcane ethanol production. sugar, such as bagasse and straw, and chips and waste materials from the pulp and paper industry.
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